Ioannis Stratos

Erythropoietin Improves Functional and Histological Recovery of Traumatized Skeletal Muscle Tissue

Apart from its hematopoietic effect, erythropoietin (EPO) is known as pleiotropic cytokine with anti‐inflammatory and anti‐apoptotic properties. Here, we evaluated for the first time the EPO‐dependent regeneration capacity in an in vivo rat model of skeletal muscle trauma. A myoblast cell line was used to study the effect of EPO on serum deprivation‐induced cell apoptosis in vitro. A crush injury was performed to the left soleus muscle in 80 rats treated with either EPO or saline. Muscle recovery was assessed by analysis of contraction capacities. Intravital microscopy, BrdU/laminin double immunohistochemistry and cleaved caspase‐3 immunohistochemistry of muscle tissue on days 1, 7, 14, and 42 posttrauma served for assessment of local microcirculation, tissue integrity, and cell proliferation. Serum deprivation‐induced myoblast apoptosis of 23.9 ± 1.5% was reduced by EPO to 17.2 ± 0.8%. Contraction force analysis in the EPO‐treated animals revealed significantly improved muscle strength with 10–20% higher values of twitch and tetanic forces over the 42‐day observation period. EPO‐treated muscle tissue displayed improved functional capillary density as well as reduced leukocytic response and consecutively macromolecular leakage over day 14. Concomitantly, muscle histology showed significantly increased numbers of BrdU‐positive satellite cells and interstitial cells as well as slightly lower counts of cleaved caspase‐3‐positive interstitial cells. EPO results in faster and better regeneration of skeletal muscle tissue after severe trauma and goes along with improved microcirculation. Thus, EPO, a compound established as clinically safe, may represent a promising therapeutic option to optimize the posttraumatic course of muscle tissue healing.

Vitamin D Increases Cellular Turnover and Functionally Restores the Skeletal Muscle after Crush Injury in Rats

Insufficient skeletal muscle regeneration after injury often impedes the healing process and is accompanied by functional deficiencies or pain. The aim of our study was to provide evidence that vitamin D improves muscle healing after muscle injury. Therefore, we used male rats and induced an injury of the soleus muscle. After crush injury, animals received either 8.3 mg/kg (332,000 IU/kg) body weight vitamin D or vehicle solution, s.c. After assessment of muscle force at days 1, 4, 14, and 42 after injury, sampling of muscle tissue served for analysis of proliferation, apoptosis, satellite cells, and prolyl-4-hydroxylase-β expression. Vitamin D application caused a significant increase in cell proliferation and a significant inhibition of apoptosis at day 4 after injury compared to control animals. The numbers of satellite cells were not influenced by the vitamin D application, but there was an increase in prolyl-4-hydroxylase-β expression, indicative of increased extracellular matrix proteins. This cellular turnover resulted in a faster recovery of contraction forces at day 42 in the vitamin D group. Current data support the hypothesis that vitamin D promotes the regenerative process in injured muscle. Thus, vitamin D treatment may represent a promising therapy to optimize recovery after injury.

Melatonin restores muscle regeneration and enhances muscle function after crush injury in rats

Abstract:  The goal of this study was to provide evidence that melatonin improves muscle healing following blunt skeletal muscle injury. For this purpose, we used 56 rats and induced an open muscle injury. After injury, all animals received either daily melatonin or vehicle solution intraperitoneally. Subsequent observations were performed at day 1, 4, 7, and 14 after injury. After assessment of fast twitch and tetanic muscle force, we analyzed leukocyte infiltration, satellite cell number, and cell apoptosis. We further quantified the expression of the melatonin receptor and the activation of extracellular‐signal‐regulated kinase (ERK). Chronic treatment with melatonin significantly increased the twitch and tetanic force of the injured muscle at day 4, 7, and 14. At day 1, melatonin significantly reduced the leukocyte infiltration and significantly increased the number of satellite cells when compared to the control group. Consistent with this observation, melatonin significantly reduced the number of apoptotic cells at day 4. Furthermore, phosphorylation of ERK reached maximal values in the melatonin group at day 1 after injury. Additionally, we detected the MT1a receptor in the injured muscle and showed a significant up‐regulation of the MT1a mRNA in the melatonin group at day 4. These data support the hypothesis that melatonin supports muscle restoration after muscle injury, inhibits apoptosis via modulation of apoptosis‐associated signaling pathways, increases the number of satellite cells, and reduces inflammation.

Osteogenic capacity of nanocrystalline bone cement in a weight-bearing defect at the ovine tibial metaphysis

The synthetic material Nanobone® (hydroxyapatite nanocrystallines embedded in a porous silica gel matrix) was examined in vivo using a standardized bone defect model in the ovine tibial metaphysis. A standardized 6 × 12 × 24-mm bone defect was created below the articular surface of the medial tibia condyles on both hind legs of 18 adult sheep. The defect on the right side was filled with Nanobone®, while the defect on the contralateral side was left empty. The tibial heads of six sheep were analyzed after 6, 12, and 26 weeks each. The histological and radiological analysis of the defect on the control side did not reveal any bone formation after the total of 26 weeks. In contrast, the microcomputed tomography analysis of the defect filled with Nanobone® showed a 55%, 72%, and 74% volume fraction of structures with bone density after 6, 12, and 26 weeks, respectively. Quantitative histomorphological analysis after 6, and 12 weeks revealed an osteoneogenesis of 22%, and 36%, respectively. Hematoxylin and eosin sections demonstrated multinucleated giant cells on the surface of the biomaterial and resorption lacunae, indicating osteoclastic resorptive activity. Nanobone® appears to be a highly potent bone substitute material with osteoconductive properties in a loaded large animal defect model, supporting the potential use of Nanobone® also in humans.

Fibroblast Growth Factor-2–Overexpressing Myoblasts Encapsulated in Alginate Spheres Increase Proliferation, Reduce Apoptosis, Induce Adipogenesis, and Enhance Regeneration Following Skeletal Muscle Injury in Rats

The fibroblast growth factor 2 (FGF-2) is known as pleiotropic cytokine with myoblast proliferative properties. In the present study, we tested the hypothesis that gene transfer of human FGF-2 via transplantation of genetically modified L8-myoblast encapsulated in alginate modulates the skeletal muscle recovery after crush injury in Wistar rats. Therefore, we performed a crush injury to the soleus muscle and transplanted alginate spheres containing myoblasts genetically modified to overexpress human FGF-2 (FGF-2) or a luciferase (LUC) cDNA at the site of injury. Animals that underwent muscle injury without transplantation of alginate spheres served as control (control). At day 4 after trauma the FGF-2 group showed significant higher mean values of cell proliferation (bromodeoxyuridine immunohistochemistry) and significant lower values of cell apoptosis (terminal deoxynucleotidyl transferase nick end labeling histology) compared to animals receiving luciferase-overexpressing myoblasts. At the same time point adiponectin expression (ACRP30 immunohistochemistry) was increased in the FGF-2 group exclusively. The p75(NTR) expression (p75(NTR) immunohistochemistry) significantly improved in both the FGF-2 and LUC group compared to the control group. Functional analysis of the injured muscle did not reveal a significant increase of the muscle force in the FGF-2 group compared to the control and LUC group 14 days after injury. In vitro analysis for 14 days of the FGF-2-modified spheres demonstrated at day 7 and day 14 a significant increase of the relative cell count as well as of the relative viable cell count in the FGF-2 myoblast spheres compared to luciferase myoblast spheres. Additionally, the expression of FGF-2 (enzyme-linked immunosorbent assay analysis) and luciferase (chemiluminescence analysis) persisted in vitro for 4 and 14 days, respectively. These results demonstrate that FGF-2-overexpressing myoblasts cannot considerably improve muscle strength but are able to modulate the proliferation as well as the apoptosis of injured muscle tissue mainly by conducting adipogenesis.

Open blunt crush injury of different severity determines nature and extent of local tissue regeneration and repair.

Insufficiency of skeletal muscle regeneration is often accompanied with functional deficiencies. The goal of our study was to assess the restoration of peripheral muscle upon injury of different severity. Blunt crush injury of the soleus muscle in rats was induced by a clamp and stepwise amplified in severity by rising the locking level of the clamp, resulting in three different groups (1× lock; 2× lock; 3× lock; n = 30 animals per group). After assessment of the fast twitch and tetanic contraction capacity at days 1, 4, 7, 14, and 42 postinjury sampling of muscle tissue served for analysis of cell proliferation, including satellite cells, apoptosis, and leukocyte infiltration. Contraction force analysis demonstrated significantly higher values of relative muscle strength in the 1× lock group compared to the two other groups over 42 days. Calculation of the twitch‐to‐tetanic force ratio revealed significantly higher mean values at days 1, 7, and 14 in the animals of group 2× lock and 3× lock, indicating a transformation toward a fast‐twitching muscular phenotype. Moreover, cell proliferation during the first 4 days was found dependent on the severity of muscle injury in that the higher the severity the higher the proliferation. At the same time, cell apoptosis was found increased, and at day 1 the local leukocyte infiltration was significantly higher in the 3× lock compared to the 1× lock group. These data indicate that severity of injury correlates with local repair responses, which, however, are not necessarily sufficient to fully restore muscle function.

Erythropoietin enhances the regeneration of traumatized tissue after combined muscle-nerve injury.

BACKGROUND Erythropoietin (EPO) is a pleiotropic cytokine with neuroprotective, anti-inflammatory, and muscle regenerative properties. The purpose of our study was to analyze the regenerative capacity of systemically applied EPO in a combined muscle-nerve injury model. METHODS We performed a crush injury to the left soleus muscle in 84 male Wistar rats. Using an instrumented clamp, the muscle was crushed over its complete length. Simultaneously, the ipsilateral sciatic nerve was sham manipulated or crushed. Upon induction of the trauma, animals received either EPO (E) (single application of 5,000 IU/kg body weight intraperitonial) or vehicle solution (K). After in vivo assessment of mechanical pain according to Frey, thermal hyperalgesia, latency of nerve conduction velocity, and strength of the soleus muscle were analyzed at days 1, 7, and 42 postinjury (n = 7 per group). Cell proliferation and apoptosis were assessed by means of histology and immunohistochemistry. RESULTS Combined muscle-nerve injury showed a significant loss of muscle strength, which incompletely recovered within 42 days. Rats treated with EPO showed an increased muscle strength after 7 days and 42 days compared with the control group. Pain behavior was highest in the muscle-nerve injured animals at day 7. EPO decreased the pain and increased nerve conduction velocity. Nerve injury diminished proliferation of muscle cells, whereas simultaneous therapy with EPO resulted in a boost of bromdesoxyuridine-positive cells. CONCLUSIONS EPO promoted muscle restoration and enhanced nerve recovery after combined muscle-nerve injury. Thus, EPO might represent an attractive therapeutic option to optimize the posttraumatic course after injury.

Compartmental and muscular response to closed soft tissue injury in rats investigated by oxygen-to-see and intravital fluorescence microscopy.

BACKGROUND Closed soft tissue injury (CSTI) induces local inflammation and progressive microvascular dysfunction. The aim of the study was to evaluate and compare the microvascular changes systematically in a precompartmental tissue injury by oxygen-to-see (O2C), a combined laser Doppler flowmetry and spectrophotometry system, and intravital fluorescence microscopy (IVM). METHODS Fourteen Wistar rats were subjected to a trauma and a control group (both n = 7). CSTI was performed on the left lower limb by means of a standardized impact device. Controls received a sham CSTI. Capillary blood flow (QRBC), oxygen saturation (sO2), and postcapillary filling pressure (rHb) were measured noninvasively by O2C assessed in 2-mm and 8-mm depth underneath the skin. Measurements were done before and after trauma and hourly up to 24 hours. IVM of the soleus muscle was performed after 24 hours. RESULTS Before CSTI, O2C parameters did not reveal a difference between both groups. Up to 2 hours after trauma, QRBC was significantly increased in 8-mm tissue depth. No significant changes of sO2 and rHb were noted compared with controls. In 2-mm depth, significantly reduced QRBC and rHb levels were observed compared with 8 mm but with no significant changes after CSTI. IVM showed a significant increase of postcapillary blood flow with decreased functional capillary density, increased macromolecular leakage, and increased nicotinamide adenine dinucleotide hydride. Conclusions After CSTI in rats, there was an immediate increase of compartmental capillary blood flow with a slight increase of muscle oxygen saturation and unchanged postcapillary venous filling pressures as sign of a redistribution of blood between soft and muscle tissue. The severity of pathologic changes in the compartment was not reflected by O2C but by IVM.

Sprint Interval Training Induces A Sexual Dimorphism but does not Improve Peak Bone Mass in Young and Healthy Mice

Elevated peak bone mass in early adulthood reduces the risk for osteoporotic fractures at old age. As sports participation has been correlated with elevated peak bone masses, we aimed to establish a training program that would efficiently stimulate bone accrual in healthy young mice. We combined voluntary treadmill running with sprint interval training modalities that were tailored to the individual performance limits and were of either high or intermediate intensity. Adolescent male and female STR/ort mice underwent 8 weeks of training before the hind legs were analyzed for cortical and trabecular bone parameters and biomechanical strength. Sprint interval training led to increased running speeds, confirming an efficient training. However, males and females responded differently. The males improved their running speeds in response to intermediate intensities only and accrued cortical bone at the expense of mechanical strength. High training intensities induced a significant loss of trabecular bone. The female bones showed neither adverse nor beneficial effects in response to either training intensities. Speculations about the failure to improve geometric alongside mechanical bone properties include the possibility that our training lacked sufficient axial loading, that high cardio-vascular strains adversely affect bone growth and that there are physiological limits to bone accrual.

Muscle, Ligament and Tendon Regeneration

Muscle injury and degenerative muscle diseases are disabling conditions that are currently challenging orthopedic surgeons, neurologist and specialists in rehabilitative medicine. Upon traumatic or degenerative changes in the structure of the muscle, regeneration befalls mainly by increased proliferation of satellite cells. If the injury is extensive fibrosis and scar tissue formation occurs. Till now various alternative therapeutic ways have been proposed to boost muscle regeneration. These methods include the use of growth factors, antioxidative therapeutic approaches, cell based therapy and cell transplantation as well as the use of scaffolds. Growth factors, antioxidative substances and endogenous polypeptides can not only influence but also control the natural repair processes by acting on different intracellular pathways. Cell orientated therapies have been popular in muscle regeneration mainly because small quantities of cells are needed to achieve therapeutic effects. Transplantation of stem cells, myoblasts or genetically modified cells, have been used after injury to restore muscle structure and function. Furthermore, scaffolds have been used to repair muscle defects and to generate new muscle fibers.